Structure and function of GluN1-3A NMDA receptor excitatory glycine receptor channel

N-methyl-d-aspartate receptors (NMDARs) and other ionotropic glutamate receptors (iGluRs) mediate most of the excitatory signaling in the mammalian brains in response to the neurotransmitter glutamate. Uniquely, NMDARs composed of GluN1 and GluN3 are activated exclusively by glycine, the neurotransmitter conventionally mediating inhibitory signaling when it binds to pentameric glycine receptors. The GluN1-3 NMDARs are vital for regulating neuronal excitability, circuit function, and specific behaviors, yet our understanding of their functional mechanism at the molecular level has remained limited. Here, we present cryo–electron microscopy structures of GluN1-3A NMDARs bound to an antagonist, CNQX, and an agonist, glycine. The structures show a 1-3-1-3 subunit heterotetrameric arrangement and an unprecedented pattern of GluN3A subunit orientation shift between the glycine-bound and CNQX-bound structures. Site-directed disruption of the unique subunit interface in the glycine-bound structure mitigated desensitization. Our study provides a foundation for understanding the distinct structural dynamics of GluN3 that are linked to the unique function of GluN1-3 NMDARs.

The PDF file includes: Figs. S1 to S6 Table S1 Legend for movie S1 Other Supplementary Material for this manuscript includes the following:

Fig. S1 .
Fig. S1.Construct design and purification of GluN1a-3A receptors.(A) Schematic representation of GluN1a (magenta) and GluN3A (grey) depicting their modular structures, including the amino terminal domain (ATD, top), ligand binding domain (LBD, middle), and transmembrane domain (TMD, bottom).The C-terminal domain of GluN3A was truncated by 148 amino acids and terminated with Ser967, and the GluN1a subunit was truncated by 91 amino acids and terminated with Gln847.(B) SDS-PAGE gel of purified GluN1a-3A ΔCTD receptors with or without the stabilizing inter-subunit disulfide bridge formed by the GluN1a Phe810Cys and GluN3A Thr675Cys mutations.Proteins were mixed with sample buffer supplemented with or without 50 mM DTT, resolved by SDS-PAGE, and stained with Coomassie.Presence of a ~200 kDa band suggests formation of the inter-subunit crosslink.(C) Size exclusion chromatography of the GluN1a ΔCTD Phe810Cys -GluN3A ΔCTD Thr675Cys receptor.Proteins were purified over Streptactin Sepharose, exchanged into PMAL-8, and resolved on a Superose 6 10/300 column monitoring UV.Fractions from the main peak (arrow 2) representing monodisperse NMDARs were used for cryo-EM studies.Additional peaks indicated by arrows represent aggregated protein (arrow 1 and Void) and free unbound CNQX.

Fig. S2 .
Fig. S2.Electrophysiological characterization of GluN1a-3A constructs.(A) Quantification of experiments performed in Figure 1.Peak currents before and after treatment with CNQX or CGP-78608 were plotted (left panels).Bar graphs measure the extent of inhibition by CNQX, or the fold potentiation upon CGP-78608 treatment.Bars represent the mean, each point represents a single patch, and error bars represent the SEM.(B) CNQX inhibition of WT GluN1a-3A and the cryo-EM construct under the tonic presence of CGP-78608.Patches were held in 500 nM CGP-78608 and glycine triggered currents were measured before and after a 10 s treatment with CNQX (left).

Fig. S3 .
Fig. S3.Particle processing and cryo-EM data of GluN1a-3A in the presence of CNQX.(A) Micrographs and particles were initially processed on-the-fly to monitor ice quality and particle behavior.2D averages were generated and used for template picking from micrographs processed separately in cryoSPARC.(B) Local resolution map of the final reconstruction.(C) Fourier shell correlations of the CNQX-bound receptor.

Fig. S4 .
Fig. S4.Representative fit of CNQX-bound GluN1a-3A to the cryo-EM density.The cartoon model for GluN1a (chains A and C) are shown in magenta and cryo-EM density is shown as blue mesh.The cartoon model for GluN3A (chains B and D) are shown in grey.The overall fit of each respective chain is shown at different magnifications from the entire chain (left), a cross section of the LBDs (middle), and two examples of per-residue fit within the LBDs (right).

Fig. S5 .
Fig. S5.Particle processing and cryo-EM data of cryo-EM data of GluN1a-3A with glycine.(A) Micrographs and particle processing.Preliminary on-the-fly processing was used to generate 2D classes for template picking in cryoSPARC.An initial reconstruction from these picks was used to generate 2D classes for a second iteration of template picking.Multiple rounds of heterogeneous refinement and ab initio were used to clean the particles.(B and C) Local resolution of the overall glycine-bound GluN1-3A volume and the local refinement of the LBD heterotetramer.(D and E) Fourier shell correlations of the cryo-EM data and model validation.

Fig. S6 .
Fig. S6.Representative fit of glycine-bound GluN1a-3A to the cryo-EM density.The cartoon model for GluN1a (chains A and C) are shown in magenta and cryo-EM density is shown as blue mesh.The cartoon model for GluN3A (chains B and D) are shown in grey.The overall fit of each respective chain is shown at different magnifications from the entire chain (left), a cross section of the LBDs (middle), and two examples of per-residue fit within the LBDs (right).